Reduction and refinement of murine models of bacterial infection

Aims

This research aimed to reduce the use of animals in the investigation of clinically-relevant bacterial diseases by developing and implementing a method of monitoring infection using bioluminescence.

Background

Infectious diseases are a major cause of mortality and morbidity worldwide. Mice are often used to investigate disease pathogenesis and to develop treatments. Traditionally, infected animals are killed at defined time points and tissues removed to determine localisation and degree of infection, and response to treatment; a six time point experiment would use up to 36 animals. Non-invasive detection of bacteria within live animals enables longitudinal monitoring and reduces the number of mice used by avoiding culling cohorts at various time points and allowing each animal to act as its own control.

Research details and methods

Plasmid vectors were developed for expression of luciferase in the targeted bacteria.  Infection and disease progression were monitored in real time in mice by bioluminescence imaging.

Key impacts and findings

The project developed a clinically-relevant bioluminescent strain of Streptococcus pyogenes, a Gram positive bacterium associated with a range of human diseases. By monitoring the bioluminescence, infected mice could be studied for around four days without the need to cull groups at pre-determined time points. This allowed the number of animals to be reduced by four-fold. The technique also provided more precise quantitation of the bacterial infectious dose, and revealed previously unknown niches of infection. The ability to identify mice in which the disease was progressing rapidly enabled the use of improved humane endpoints to minimise suffering. 

Related Content

 

According to WHO estimates, in 2001, infectious diseases caused 14.7 million deaths – 26% of global mortality. Murine models play an essential role in the race to develop improved vaccines and therapeutic agents but are limited by the need to use large numbers of animals to obtain quantitative microbiological data.

Bioluminescence imaging (BLI) of microorganisms during infection exploits a highly sensitive, non-toxic analytical technique based on the detection of visible light produced by luciferase-catalysed reactions. BLI allows the non-invasive detection of live cells from within intact living animals in real-time. Multiple imaging of the same animal throughout an experiment allows disease progression to be followed with extreme accuracy, while allowing each animal to act as its own control. Furthermore, when constitutively expressed, bioluminescence is related to bacterial numbers and can therefore be used for quantification of pathogen burden, allowing for humane euthanasia perhaps even before the onset of clinical symptoms. The use of BLI has resulted in important new insights into the niches exploited by pathogens during infection, challenging conventional dogma and opening new avenues for research into therapeutic agents and vaccines. In this project we wish to overcome some of the obstacles to implementing BLI for infectious diseases research by constructing a suite of vectors suitable for developing dual bioluminescent/fluorescent derivatives in a wide range of both Gram-positive and Gram-negative bacteria. Importantly, even in those institutions which do not have access to BLI equipment, the ability to measure bacterial numbers in real time by luminometry will enable researchers to ensure that animals are given the correct infectious dose, reducing variation between experiments and the risk of unexpected adverse effects. In addition, we wish to develop dual bioluminescent/fluorescent derivatives of the important human pathogen Group A Streptococcus (GAS) which causes immense human morbidity and mortality and utilise BLI to interrogate and refine existing murine models of GAS infection.

GAS strains produce a range of virulence factors and an array of disease manifestations, including tonsillitis, skin infections, sepsis, necrotising fasciitis, and toxic shock syndrome.

Numerous murine models of GAS infection are used; ranging from non-lethal representations of colonisation and acute infections to severe invasive disease with up to 100% mortality within 24-96 hours. BLI will aid in addressing numerous outstanding questions of GAS pathogenicity and a number of the murine models used in GAS research would greatly benefit from the implementation of more humane endpoints.

Alam FM, Bateman C, Turner CE, Wiles S, Sriskandan S (2013) Non-invasive monitoring of Streptococcus pyogenes vaccine efficacy using biophotonic imaging. PLoS ONE 8(11): e82123. doi:10.1371/journal.pone.0082123

Alam FM, Turner CE, Smith K, Wiles S, Sriskandan S (2013) Inactivation of the CovR/S virulence regulator impairs infection in an improved murine model of Streptococcus pyogenes naso-pharyngeal  infection. PLoS ONE 8(4): e61655 doi:10.1371/journal.pone.0061655

Andreu N, Zelmer A, Wiles S (2011) Non-invasive biophotonic imaging for studies of infectious disease. FEMS Microbiology Reviews 35(2): 360-394. doi:10.1111/j.1574-6976.2010.00252.x

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Project grant

Status:

Closed

Principal investigator

Professor Shiranee Sriskandan

Institution

Imperial College London

Co-Investigator

Dr Siouxsie Wiles

Grant reference number

G0800720/1

Award date:

Jan 2009 - Mar 2011

Grant amount

£276,759